Recruitment of tumor necrosis factor receptor-associated factor family proteins to apoptosis signal-regulating kinase 1 signalosome is essential for oxidative stress-induced cell death

Apoptosis signal-regulating kinase 1 (ASK1) plays a pivotal role in oxidative stress-induced cell death. Reactive oxygen species disrupt the interaction of ASK1 with its cellular inhibitor thioredoxin and thereby activates ASK1. However, the precise mechanism by which ASK1 freed from thioredoxin undergoes oligomerization-dependent activation has not been fully elucidated. Here we show that endogenous ASK1 constitutively forms a high molecular mass complex including Trx ( approximately 1,500-2,000 kDa), which we designate ASK1 signalosome. Upon H(2)O(2) treatment, the ASK1 signalosome forms a higher molecular mass complex at least in part because of the recruitment of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. Consistent with our previous findings that TRAF2 and TRAF6 activate ASK1, H(2)O(2)-induced ASK1 activation and cell death were strongly reduced in the cells derived from Traf2-/- and Traf6-/- mice. A novel signaling complex including TRAF2, TRAF6, and ASK1 may thus be the key component in oxidative stress-induced cell death.     

Cigarette smoke extract induces endothelial cell injury via JNK pathway

Cigarette smoking is the most crucial factor responsible for chronic obstructive pulmonary disease (COPD). The precise mechanisms of the development of the disease have, however, not been fully understood. Recently, impairment of pulmonary endothelial cells has been increasingly recognized as a critical pathophysiological process in COPD. To verify this hypothesis, we examined how cigarette smoke extract (CSE) damages human umbilical vein endothelial cells (HUVECs). CSE activated c-Jun N-terminal kinase (JNK), and treatment of HUVECs with SP600125, a specific inhibitor of the JNK pathway, significantly suppressed endothelial cell damage by CSE. In contrast, inhibition of the extracellular-regulated kinase or the p38 pathway did not affect the cytotoxicity of CSE. Furthermore, anti-oxidants superoxide dismutase and catalase reduced CSE-induced JNK phosphorylation and endothelial cell injury. These results indicate that CSE damages vascular endothelial cells through the JNK pathway activated, at least partially, by oxidative stress.     

Attenuated phosphorylation of heat shock protein 27 correlates with tumor progression in patients with hepatocellular carcinoma

Heat shock protein 27 (HSP27) is expressed at high levels in human hepatocellular carcinoma (HCC). We examined correlations of total HSP27 and serine phosphorylated (Ser-15, Ser-78, and Ser-82) HSP27 levels in HCC tissues with clinical and pathologic characteristics in 48 resected HCC specimens. The levels of total and Ser-phosphorylated HSP27 were evaluated by Western blot analysis. Immunohistochemical analysis of HSP27 expression was also performed on some samples. Phosphorylation of HSP27 was detected in all 48 HCC tissues. Levels of phosphorylated HSP27 were correlated inversely with tumor size, microvascular invasion of HCC, and tumor stage by TNM classification. In contrast, only microvascular invasion showed an inverse correlation with total HSP27 levels. The decrease in phosphorylated HSP27 in progressed HCC was also observed by immunohistochemistry. Levels of phosphorylated HSP27 gradually decreased in parallel with HCC progression. Our findings suggest that phosphorylated HSP27 may have a suppressive role in progression of human HCC.     

Macrophage colony-stimulating factor induces vascular endothelial growth factor production in skeletal muscle and promotes tumor angiogenesis

Although M-CSF has been used for myelosuppression due to chemotherapy in patients with solid tumors, the effect of exogenous M-CSF on tumor angiogenesis has not been studied. In this study we showed that M-CSF has the ability to accelerate solid tumor growth by enhancing angiogenesis with a novel mechanism. M-CSF accelerated intratumoral vessel density in tumors inoculated into mice, although it did not accelerate the proliferation of malignant cells and cultured endothelial cells in vitro. In both the absence and the presence of tumors, M-CSF significantly increased the circulating cells that displayed phenotypic characteristics of endothelial progenitor cells in mice. Moreover, M-CSF treatment induced the systemic elevation of vascular endothelial growth factor (VEGF). VEGFR-2 kinase inhibitor significantly impaired the effect of M-CSF on tumor growth. In vivo, M-CSF increased VEGF mRNA expression in skeletal muscles. Even after treatment with carageenan and anti-CD11b mAb in mice, M-CSF increased VEGF production in skeletal muscles, suggesting that systemic VEGF elevation was attributed to skeletal muscle VEGF production. In vitro, M-CSF increased VEGF production and activated the Akt signaling pathway in C2C12 myotubes. These results suggest that M-CSF promotes tumor growth by increasing endothelial progenitor cells and activating angiogenesis, and the effects of M-CSF are largely based on the induction of systemic VEGF from skeletal muscles.     

Suppression of the caspase cleavage of beta-amyloid precursor protein by its cytoplasmic phosphorylation

beta-Amyloid precursor protein (APP) is a type I transmembrane protein. Its cleavages by beta- and gamma-secretases yield beta-amyloid, which is the main constituent of senile plaques in Alzheimer's disease (AD). In apoptotic cells and AD brains, APP is alternatively cleaved by caspases in the cytoplasmic region after the Asp664 residue (with respect to the numbering conversion for the APP695 isoform). Caspase-cleaved fragments of APP are cytotoxic and have been implicated in AD pathogenesis; however, the mechanisms regulating the cleavage have not been studied. APP is constitutively phosphorylated at Thr668 in brain. In the present study, we demonstrate that APP phosphorylated at Thr668 is less vulnerable to cytoplasmic cleavage by caspase-3 and caspase-8. This suggests that APP phosphorylation suppresses the generation of caspase-cleaved fragments of APP in the brain and that perturbation of this phosphorylation may be involved in APP-mediated neurotoxicity.     

Singlet oxygen inhibits the repair of photosystem II by suppressing the translation elongation of the D1 protein in Synechocystis sp. PCC 6803

Singlet oxygen, generated during photosynthesis, is a strong oxidant that can, potentially, damage various molecules of biological importance. We investigated the effects in vivo of singlet oxygen on the photodamage to photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. Increases in intracellular concentrations of singlet oxygen, caused by the presence of photosensitizers, such as rose bengal and ethyl eosin, stimulated the apparent photodamage to PSII. However, actual photodamage to PSII, as assessed in the presence of chloramphenicol, was unaffected by the production of singlet oxygen. These observations suggest that singlet oxygen produced by added photosensitizers acts by inhibiting the repair of photodamaged PSII. Labeling of proteins in vivo revealed that singlet oxygen inhibited the synthesis of proteins de novo and, in particular, the synthesis of the D1 protein. Northern blotting analysis indicated that the accumulation of psbA mRNAs, which encode the D1 protein, was unaffected by the production of singlet oxygen. Subcellular localization of polysomes with bound psbA mRNAs suggested that the primary target of singlet oxygen might be the elongation step of translation.     

Fatty acids regulate pigmentation via proteasomal degradation of tyrosinase: a new aspect of ubiquitin-proteasome function

Fatty acids are common components of biological membranes that are known to play important roles in intracellular signaling. We report here a novel mechanism by which fatty acids regulate the degradation of tyrosinase, a critical enzyme associated with melanin biosynthesis in melanocytes and melanoma cells. Linoleic acid (unsaturated fatty acid, C18:2) accelerated the spontaneous degradation of tyrosinase, whereas palmitic acid (saturated fatty acid, C16:0) retarded the proteolysis. The linoleic acid-induced acceleration of tyrosinase degradation could be abrogated by inhibitors of proteasomes, the multicatalytic proteinase complexes that selectively degrade intracellular ubiquitinated proteins. Linoleic acid increased the ubiquitination of many cellular proteins, whereas palmitic acid decreased such ubiquitination, as compared with untreated controls, when a proteasome inhibitor was used to stabilize ubiquitinated proteins. Immunoprecipitation analysis also revealed that treatment with fatty acids modulated the ubiquitination of tyrosinase, i.e. linoleic acid increased the amount of ubiquitinated tyrosinase whereas, in contrast, palmitic acid decreased it. Furthermore, confocal immunomicroscopy showed that the colocalization of ubiquitin and tyrosinase was facilitated by linoleic acid and diminished by palmitic acid. Taken together, these data support the view that fatty acids regulate the ubiquitination of tyrosinase and are responsible for modulating the proteasomal degradation of tyrosinase. In broader terms, the function of the ubiquitin-proteasome pathway might be regulated physiologically, at least in part, by fatty acids within cellular membranes.     

Contribution of bone-marrow-derived cells to choroidal neovascularization

We investigated the involvement of bone-marrow derived cells to experimental choroidal neovascularization (CNV) in mice, whose bone marrow was reconstituted by either unfractionated bone-marrow cells or Lin-c(-)Kit(+)Sca-1+ enriched presumable hematopoietic stem cells from the green fluorescent protein (GFP) transgeneic mice. Immunohistochemical analysis demonstrated the presence of GFP-positive cells in the CNV lesion after unfractionated bone-marrow transplantation, as well as Lin-c(-)Kit(+)Sca-1+ cell transplantation. Some of the GFP-expressing cells also expressed CD-31 and PanEC antigen, markers of vascular endothelial cells. Our results suggest that bone-marrow derived cells may contribute endothelial cells in CNV.